The present disclosure relates to methods for preserving and storing biological specimens, such as anaerobe microorganisms. Compositions for use in such methods are also disclosed. This preserves the specimens for later usage, and also enables recovery of the stored specimens at numbers near those initially placed in storage.
Temperature is a key parameter for storing biological specimens such as cells, tissues, and particularly anaerobe microorganisms. Specimens are usually stored in some form of container (box, tube, etc.) that is kept at room temperature, cold temperatures (refrigerated at 5-8 degrees Celsius), or frozen temperatures (sub-zero, i.e. below 0 degrees Celsius). Frozen temperatures are ordinarily about minus twenty (−20) degrees Celsius, while frozen temperatures suitable for cryogenic storage are usually about minus seventy (−70) degrees Celsius or lower. Generally, the colder the temperature, the longer the intended storage time for the specimens. Freezing or sub-zero temperatures reduce the occurrence of chemical reactions, producing a static condition, which in turn acts to protect the specimens from damage during storage.
Freezing has evolved as a method of choice, and cryo-freezing in particular has become the most used storage temperature range for biological materials. However, some problems can occur in freezing biological materials. The most cited problem is ice formation. Since biological materials are high in water content (about 80% on average), the formation of ice crystals is almost inevitable. Ice crystals are thought to produce physical damage to cells and tissues. Cell damage mostly occurs during the freezing step, although damage can occur during thawing as well. Damage, as measured by cell death of the original cell population to be frozen, may range from 20% to over 90% depending upon methods and materials used and the nature of the biological material. This damage may be significant to the point that any survivors of freezing may constitute a small part of the initial population so as to not be representative of that population.
Another way to store biological materials is to employ freeze drying, or lyophilization. This method involves bringing the material to freezing temperatures while removing moisture. The end product is a dried powder.
After restoring the lyophile, the recovered portion of viable cells can be very low compared to the initial cell population that was preserved, e.g. less than 1:100 of cells (recovered:initial) are recovered (i.e. 1%). In some cases, the recoverable portion of cells may be only one cell per million initial cells, or less. This can raise the question of whether the recovered cells are truly representative of the initially frozen population. Thus, preservation/storage methods that retain a higher percentage of the initial population are favored.
One way to prevent or limit ice crystal damage to tissue is to bring the material to a “glass” state in which ice crystals are not formed. In the glass state, ice is considered to be an amorphous solid in which the water molecules are arranged randomly compared to “regular” ice, in which the water molecules are arranged in a hexagonal lattice. The production of amorphous ice depends on a fast rate of cooling to about −137 degrees Celsius. If ice crystals are not formed, then freezing damage does not occur.